In recent years, reagents utilizing enzyme reactions have broadly been used for inspecting and diagnosing morbid states, instead of conventional chemical reagents.
Each of these enzyme reaction-aided reagents utilizes the properties of enzymes to convert a specific component in the living body into a detectable substance. In general, in such an assay system, a substance (A) to be measured is converted into an intermediate product (I-1) using an enzyme (a) specific for the substance, further converted into another intermediate product (I-2) by allowing another enzyme (i-1) specific for the intermediate product (I-1) to exert its action and, by repeating such reactions, the substance (A) to be measured is ultimately converted into a detectable substance (F). Then, the detectable substance (F) is determined based on changes in color tone measured with a spectrophotometer or a fluorophotometer, or with the naked eye. ##STR1##
In such reagents, NAD and its reduced form (NADH), its analogous compound NADP and its reduced form (NADPH), are broadly used as the detectable substance (F) (these compounds hereinafter are generally referred to as "nicotine nucleotides"). Since changes in absorbance at an ultraviolet region (around 340 nm) are observed in these nicotine nucleotides, reagents have been proposed in which the formation or decrease of nicotine nucleotides is measured using a spectroscope. Also, a large number of reports have been published on the use of reagents characterized in that, by reacting diaphorase with the NADH or NADPH thus formed, tetrazolium is converted into formazan and measured at a visible range (for example, bile acid (JP-A-60-214900, and Rinsyo Kagaku (Japanese Journal of Clinical Chemistry), vol. 19, pp. 290-299 (1990); the term "JP-A" as used herein means an "unexamined published Japanese patent application"), triglyceride (JP-A-55-14899), alcohol (JP-B-4-3947; the term "JP-B" as used herein means an "examined Japanese patent publication), amylase (JP-B-63-37640), creatine kinase (JP-A-58-16699 and JP-B-4-70000), polyamine (JP-B-6-68490), glucose (JP-B-7-34757) and benzylamine (JP-A-7-184693)).
In addition, in order to improve the measuring accuracy of nicotine nucleotides, several reagents have been proposed in which resazulin is converted into fluorescence-developing resorufine by the action of diaphorase, which is measured based on its fluorescence strength. For example, a reagent for measuring alanine prepared by dissolving NAD, resazulin sodium salt, diaphorase and alanine dehydrogenase in a carbonate buffer of pH 9.0 Journal of Biochemistry, vol. 96, pp. 1-8 (1984)! and a reagent for measuring bile acid prepared by dissolving 3.alpha.--OH steroid dehydrogenase, NAD, resazulin sodium salt and diaphorase in a buffer of pH 3 to 13 (JP-B-56-39199) have been proposed.
However, because an adequate quantitative evaluation cannot be obtained by such reagents which employ a fluorescence-developing substance, a method has been proposed in which a sample containing bile acid as a substance to be measured is treated at high temperature (for example at 67.degree. C. for 20 minutes) and then measured using the aforementioned reagent, in order to improve its quantitative analytic performance JP-B-56-39119 and Rinsyo Kagakushi (Japanese Journal of Clinical Chemistry), vol. 4, pp. 312-318 (1976)!. It has been pointed out, however, that even such a method has a bile acid recovery ratio of only about 90%. In addition, due to the use of a fluorescent substance in the measurement, impurities in the samples and reagents thus used tend to interfere in the measurement. Therefore, there is a large deviation in the measured value (JP-B-59-13197).